Modular PKS enzymes are large, multi-subunit enzyme complexes that perform the biosynthesis of polyketide secondary metabolites. See O'Hagan, D., 1991 (a full citation of any reference referred to herein by last name of first author and year of publication is located at the end of this section). Examples of polyketides made by modular PKS enzymes include the antibiotic erythromycin, the immunosuppressant FK506, and the antitumor compound epothilone. See also PCT patent publication No. 93/13663 (erythromycin); U.S. Pat. No. 6,303,342 B1 (epothilone); U.S. Pat. No. 6,251,636 B1 (oleandolide); PCT publication WO 01/27284 A2 (megalomicin); U.S. Pat. No. 5,098,837 (tylosin); U.S. Pat. No. 5,272,474 (avermectin); U.S. Pat. No. 5,744,350 (triol polyketide); and European patent publication No. 791,656 (platenolide), each of which is incorporated herein by reference. A large interest in these enzyme systems lies in the ability to manipulate the specificity or sequence of reactions catalyzed by PKSs to produce novel therapeutic compounds. See McDaniel, R., et al., 2001, and Weissman, K. J et al. 2001. A number of plasmid-based heterologous expression systems have been developed for the engineering and expression of PKSs, including multiple-plasmid systems for combinatorial biosynthesis. See McDaniel, et al., 1993, Xue, et al., 1999, and Ziermann, et al., 2000, and U.S. Pat. Nos. 6,033,883 and 6,177,262; and PCT publication Nos. 00/63361 and 00/24907, each of which is incorporated herein by reference.
In modular PKSs, active sites called “domains” are arranged in groups called “modules” that perform a single round of polyketide chain extension and modification (FIG. 1). PKS modules are typically between ˜3.5–7 kb, depending on the number of actives sites present in the module. Frequently the homology between similar active site domains (e.g. ketosynthase (KS), acyltransferase (AT), or ketoreductase (KR)) of a cognate PKS is greater than between domains of heterologous PKSs. Many sequenced PKS gene clusters contain at least two domains in which the DNA sequence identity is greater than 99% over significant lengths of nucleotide bases (i.e. >500 bp). For example, the KR and acylcarrier protein (ACP) domains from modules 2 and 5 of the oleandomycin PKS (see Shah et al., 2000, Swan, D. G., et al., 1994, and U.S. Pat. No. 6,251,636, incorporated herein by reference) each contain a 1,211 bp contiguous segment with 100% identity. In the tylosin PKS (see DeHoff et al., 1996), three 2,013–2,290 bp fragments from the KS and AT domains of modules 1, 4, and 6 all share a sequence identity greater than 99.5%. These repetitive sequences most likely arise from gene duplications or gene conversion during the evolution of the PKS. While these regions appear to be stable in the chromosome of the host organisms in which they are found, such duplications are potentially detrimental to the stable expression of plasmid-borne PKSs in hosts capable of homologous recombination.
The megalomicin 6-deoxyerythronolide B (6-dEB) synthase (meg DEBS , FIG. 1) contains duplicate regions comprising 615 bp in the KS domains and 426 bp in the AT domains of module 2 and module 6. The erythromycin 6-dEB synthase (ery DEBS) is identical in overall genetic architecture to meg DEBS (see Volchegursky, Y., et al., 2000), but does not possess any such redundant sequences. Recently, it was reported that both ery and meg DEBS produced similar yields of 6-dEB in Streptomyces lividans (see Volchegursky, Y., et al., 2000). In subsequent rounds of fermentation, titers from meg DEBS were consistently lower than those from ery DEBS. Furthermore a significant decrease in titers was observed when meg DEBS was expressed in S. coelicolor CH999, and titers could not be determined reproducibly. This titer decrease and lack of reproducible titer may relate to the regions of homology that are present in the meg but not the ery DEBS. Thus, there exists a need for methods to improve PKS genes that contain such regions of homology. The present invention provides methods and compositions to meet this and other needs.                The following articles provide background information relating to the invention and are incorporated herein by reference.            DeHoff et al. 1996. GenBank accession #U78289.    Desai et al. 2002. J. md. Microbiol. Biotech. 28:297–301.    Jacobsen et al. 1997. Science. 277:367–369.    Kao et al. 1996. Biochem. 35:12363–12368.    Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK.    Leaf et al. 2000. Biotechnol. Prog. 16:553–556.    MacNeil et al. 1992. Gene. 115:119–125.    McDaniel et al. 1993. Science. 262:1546–1557.    McDaniel et al. 2001. In Kirst et al. (ed), Enzyme technologies for pharmaceutical and biotechnological applications, p. 397–426. Marcel Dekker, Inc., N.Y.    O'Hagan, D. 1991. The polyketide metabolites. Ellis Horwood, Chichester, UK.    Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, N.Y.    Shah et al. 2000. J. Antibiotics. 53:502–508.    Swan et al. 1994. Mol. Gen. Genet. 242:358–362.    Tang et al. 2000. Chem. & Biol. 7:77–84.    Tsai et al. 1987. Mol. Gen. Genet. 208:211–218.    Volchegursky et al. 2000. Mol. Microbiol. 37:752–762.    Weissman et al. 2001. In H. A. Kirst et al. (ed.), Enzyme technologies for pharmaceutical and biotechnological applications, p. 427–470. Marcel Dekker, Inc., N.Y.    Xue et al. 1999. Proc. Natl. Acad. Sci. U.S.A. 96:11740–11745.    Ziermann et al. 2000. J. Ind. Microbiol. Biotech. 24:46–50.